The long-term objective of the research proposed here is to determine the molecular mechanisms of homologous recombination, by studying the enzymes promoting the individual steps of the process and the structures of the DNA intermediates at each step. An additional objective is to determine the mechanisms by which recombination is regulated. The research will focus on the RecBCD pathway of recombination in Escherichia coli and on meiotic recombination in Schizosaccharomyces pombe. Specific aims of the research in E. coli are to 1) verify a small RNA subunit that appears to be required for some but not all activities of the RecBCD enzyme, 2) determine the alteration in RecBCD enzyme upon cutting at Chi, resulting in loss of some but not all of its activities, 3) test a model of DNA unwinding by RecBCD enzyme, based upon the structure of the RecBCD enzyme-DNA initiation complex, and 4) analyze the structure of hybrid DNA-containing intermediates formed in the RecBCD pathway. These goals will be sought by a combination of genetic and enzymatic studies. Specific aims of the research in S. pombe are to 1) analyze meiotic recombination-deficient (rec) mutants to determine their primary defects, 2) determine the activities of the rec gene products, by cloning and sequencing the genes, purifying their products, and testing them for activities likely to be required for recombination, and 3) test the roles of enzymes, such as exonucleases I and II, likely required for recombination by generating mutants lacking these enzymes. Recombination is important for generating diversity at both the organismal and cellular levels. Aberrancies of recombination generate chromosomal rearrangements, such as deletions and translocations. Chromosomal rearrangements are associated with, and may be a cause of, birth defects and cancers. Understanding the molecular mechanisms of recombination is important in determining the causes of these diseases and possibly preventing them.